Activation of the innate immune system impairs transplant tolerance induction. Our prior work supported by the last cycle of the award, demonstrates that signaling via MyD88, an adaptor protein downstream of most Toll like receptors, prevents transplant tolerance induction. Although we determined that synergy between IL-6 and TNF-a is critical for this effect, we know neither the identity of the MyD88 expressing cell that mediates transplant tolerance resistance nor the innate activators that are sensed by this cell. It will be critical to ascertain this information for novel therapies that inhibit the innate immune response at the site of injury, i.e., within the transplant. Here we hypothesize that MyD88 expressing CD11c+ dendritic cell sense innate immune ligands, including hyaluronan to prevent transplant tolerance induction. In this proposal we will discern the nature of the innate immune ligands and the identity of the cell that responds to these ligands to induce transplant tolerance resistance. Specifically, we will use innovative genetic murine approaches to examine if MyD88 expressing CD11c+DCs are both necessary and sufficient to induce transplant tolerance resistance. In addition, we will employ both pharmacological and novel genetic approaches to deplete hyaluronan within allografts, in order to examine if hyaluronan is a critical innate ligand that induces transplant tolerance resistance. Finally, we will employ proteomic approaches to determine the identity of novel innate ligands from the lysates of immunogenic grafts that resist transplant tolerance and examine if such ligands activate CD11c+DCs via a MyD88-dependent pathway. We will also determine whether any discovered ligands are less evident in organs such as hearts that are prone to tolerance induction. Our proposed study will provide critical information concerning the cellular and molecular mechanism by which the innate immune system impairs transplant tolerance induction. Such fundamental information will help guide future endeavors at inducing transplant tolerance in the clinic, ultimately leading to novel therapies and treatment options for patients.